Gibson Calculations Page
For Gibson Master Mix, use 3.75uL in a 5uL total volume (or 7.5uL in a 10uL total volume). Calculating how much DNA to add to your Gibson Reaction: NEB recommends a total of 0.02–0.5 pmols of DNA fragments when 1 or 2 fragments are being assembled into a vector and 0.2–1.0 pmoles of DNA fragments when 4–6 fragments are being assembled. Efficiency of assembly decreases as the number or length of fragments increases. Use 75ng of Vector (50-100 ng is optimal) Based on your DNA concentration, figure out how many pmols/uL of Vector DNA you have (use promega pmol calculator). Add 2-3x the number of pmols for each of your fragments. Example: 6000bp Vector: concentration: 100ng/uL (or .1ug/uL) Using Promega: 0.025pmol/uL of DNA Reaction: Vector: 75ng x (1uL/100ng) = 0.75uL 0.75uL x (0.025pmol/uL) = 0.01875pmol of Vector 0.01875pmol of vector x 3 = 0.05625pmol of each fragment Let’s say each fragment is 500bp and 100ng/uL: Using Promega: 0.3pmol/uL of DNA 0.05625pmol x (1uL/0.3pmol) = 0.1875uL of DNA We can’t accurately pipette that small a volume, so dilute the DNA so that you are pipetting ~0.5uL of each fragment, but still adding 0.05625pmol of DNA. Here is our reaction: Vector: 0.75uL (.01875pmol) Frag A: 0.5uL (.05625pmol, which is 3xVector) after being diluted Frag B: 0.5uL (.05625pmol) after being diluted Now check your totals: Total number of pmol for entire reactions should be between 0.02-0.5pmols: our total is 0.13pmol--PERFECT! Total volume should be 2.5uL or under for a 10uL reaction and 1.25uL or under for a 5uL reaction. This volume is 1.75uL, so we will opt for the 10uL reaction: 7.5uL of Gibson Mastermix 0.75uL of Vector 0.5uL Frag A 0.5uL Frag B Water up to 10uL, so 0.75uL of water. Another Example: 25 February 2016 - LMF Gibson reaction, pET28a-StrepII-sfGFP-DmOptEnscAAs20-FLAG SITUATION: One vector, 6151 bp, and one insert, 585 bp. Vector starting concentration: 105.5 ng/uL. WANT: 75 ng of vector in reaction. Vector calculations - [] in ng/uL to [] in pmol # 75 ng x (1 uL/105 ng) = approx. 0.72 uL of vector - add this to Gibson rxn. How many pmol of vector does that mean we have? 2) Use pmol calculator online: Length of vector = 6151 bp, concentration of vector = 0.105 ng/uL. SO, picomolarity of vector = 0.026 picomoles per microliter. 3) We will add to rxn: 0.72 uL of vector x 0.026 pmol/uL = 0.0187 pmoles of vector Vector calculations - calculate from pmolarity of vector to uL of insert needed Picomoles of insert should be 2-3x picomoles of vector - we’ll go for 3x as much here, since we have only 1 insert. # 0.0187 pmoles vector x 3 =''' 0.056 pmoles of insert needed''' (**The insert was ordered directly. Need to calculate resuspension of geneblock. We received 500 ng of gene insert. If resuspend our insert in 10 uL, we will get a 50 ng/uL stock). 2) Use pmol calculator online: Length of insert = 585 bp, concentration of insert = 0.05 ug/uL. SO,' picomolarity of insert = 0.13 picomoles per microliter (pmoles/uL) ' 3) We will add to rxn: '''0.056 pmoles insert x (0.13 pmoles/uL) = 0.43 uL. ''Create table to check volumes, picomolarity:'' Volume Picomoles Vector 0.72 uL 0.0187 pmoles Insert 0.43 uL 0.056 pmoles '''TOTAL: 1.15 uL 0.0747 pmoles CHECK: ''' Our Gibson Master Mix is 1.3 x. We use 3.75 uL in a 5 uL reaction and 7.5 uL in a 10 uL reaction. Thus, total volume of DNA added CANNOT exceed 1.25 uL for a 5 uL reaction - we have 1.15 here, so this is a GO. Also, total picomoles of DNA added should be between 0.02 and 0.5 picomoles - we are 0.0747 pmoles here, so we are a GO. Let’s change the volumes slightly to get the exact 1.25 uL of DNA added. (Also, it is very hard to pipet less than 0.5 uL, so this will be better for pipetting purposes too). Volume Picomoles Vector 0.75 uL 0.0187 pmoles Insert 0.50 uL 0.056 pmoles '''TOTAL: 1.25 uL 0.0747 pmoles Check the math one more time and we are good to go!